The present invention is related to the molecular cloning of a gene encoding a flagellin protein, identified herein as FlaC, of the flagellar filament from a strain of Campylobacter.
Campylobacter jejuni is a Gram-negative spiral microaerophilic bacterium that has been recognized as a cause of secretory type diarrhea and enteritis. (Ref. 1). Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the disclosed end of the specification immediately preceding the claims. These references are hereby incorporated by reference into the present disclosure). The flagellum of C. jejuni is responsible for bacterial motility which enhances the organism""s pathogenicity. The flagellum consists of three major components; the filament, the hook, and the basal body (Ref. 2). A campylobacter cell carries a single unsheathed flagellum at one or both poles of the body. The flagella are responsible for the high motility of the organisms as aflagellate mutants are nonmotile (Refs. 3, 4, 5, 6, 7, 8, 9). A number of studies indicated that the polar flagellum plays an important role in colonization of the viscous mucus lining of the gastric intestinal tract and that it is an important virulence determinant (Refs. 3,4,7,10,11).
The basic structure of the bacterial flagellum consists of a propeller (filament) connected via a universal joint (hook) to a transmission shaft, motor and brushings (basal body) embedded in the cell envelope (Ref. 12). The flagellar filament consists of several thousand self-assembling protein (flagellin) monomers arranged in a helix. These form a hollow tube of relatively constant diameter and variable length with an over corkscrew morphology.
Most eubacterial flagellar filaments that have been characterized appear to be composed of a single kind of flagellin (Ref. 8). However a number of Eubacteria have now been shown to possess multiple flagellin genes (Refs. 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22), C jejuni (Refs. 13, 15, 17, 21) and C. coli (Refs. 6, 13) have been reported to have two flagellin genes (flaA and flaB). In C. jejuni, the flagellin genes flaA and flaB have been isolated and sequenced, however prior to the present invention a third flagellin gene had not been isolated and characterized.
It would be advantageous to provide nucleic acid molecules encoding flagellin proteins of the flagella for strains of Campylobacter and purified flagellin proteins, including flaC for use as antigens, immunogenic compositions, including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents.
The present invention is directed towards the provision of purified and isolated nucleic acid molecules encoding a flagellin protein C (FlaC) of a strain of Campylobacter or a fragment or an analog of the flagellin protein. The nucleic acid molecules provided herein are useful for the specific detection of strains of Campylobacter, and for diagnosis of infection by Campylobacter. The purified and isolated nucleic acid molecules provided herein, such as DNA, are also useful for expressing the flac gene by recombinant DNA means for providing, in an economical manner, purified and isolated FlaC proteins, subunits, fragments or analogs thereof. The FlaC protein, subunits or fragments thereof or analogs thereof, as well as nucleic acid molecules encoding the same and vectors containing such nucleic acid molecules, are useful in immunogenic compositions against diseases caused by Campylobacter, the diagnosis of infection by Campylobacter and as tools for the generation of immunological reagents. Monoclonal antibodies or mono-specific antisera (antibodies) raised against the FlaC protein produced in accordance with aspects of the present invention are useful for the diagnosis of infection by Campylobacter, the specific detection of Campylobacter (in for example in vitro and in vivo assays) and for the treatment of diseases caused by Campylobacter.
Peptides corresponding to portions of the FlaC protein or analogs thereof are useful immunogenic compositions against disease caused by Campylobacter, the diagnosis of infection by Campylobacter and as tools for the generation of immunological reagents. Monoclonal antibodies or antisera raised against these peptides, produced in accordance with aspects of the present invention, are useful for the diagnosis of infection by Campylobacter, the specific detection of Campylobacter (in, for example, in vitro and in vivo assays) and for use in passive immunization as a treatment of disease caused by Campylobacter.
In accordance with one aspect of the present invention, there is provided a purified and isolated nucleic acid molecule encoding a flagellin protein (FlaC) of flagellum of a strain of Campylobacter, more particularly, a strain of Campylobacter jejunis, or a fragment or an analog of the FlaC protein.
In one preferred embodiment of the invention, the nucleic acid molecule may encode the FlaC protein of the Campylobacter strain.
In another aspect of the present invention, there is provided a purified and isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: (a) the entire nucleotide sequence set out in FIG. 1 (SEQ ID No: 1), or the complementary sequence of said sequence; (b) the coding nucleotide sequence set out in FIG. 1 (SEQ ID No: 2), or the complementary sequence of said sequence; (c) a nucleotide sequence encoding the amino acid sequence set forth in FIG. 1 (SEQ ID No: 3); and (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c). The DNA sequence defined in (c) preferably has at least about 90% sequence identity with any one of the DNA sequences defined in (a) and (b).
In an additional aspect, the present invention includes a vector adapted for transformation of a host, comprising a nucleic acid molecule as provided herein. The vector may be one having the characteristics of plasmid pD2-2.
The plasmids may be adapted for expression of the encoded FlaC protein, fragments or analogs thereof, in a heterologous or homologous host, in either a lipidated or non-lipidated form. Accordingly, a further aspect of the present invention provides an expression vector adapted for transformation of a host comprising a nucleic acid molecule as provided herein and expression means operatively coupled to the nucleic acid molecule for expression by the host of the FlaC protein or the fragment or analog of the FlaC protein. In specific embodiments of this aspect of the invention, the nucleic acid molecule may encode substantially all the FlaC protein of the Campylobacter strain. The expression means may include a nucleic acid portion encoding a leader sequence for secretion from the host of the FlaC protein or the fragment or the analog of the FlaC protein. The expression means also may include a nucleic acid portion encoding a lipidation signal for expression from the host of a lipidated form of the FlaC protein or the fragment or the analog of the FlaC protein. The host may be selected from, for example, Escherichia coli, Bordetella, Bacillus, Haemophilus, Moraxella, fungi, yeast or baculovirus and Semliki Forest virus expression systems may be used.
In an additional aspect of the invention, there is provided a transformed host containing an expression vector as provided herein. The invention further includes a recombinant FlaC protein or fragment or analog thereof producible by the transformed host. Further aspects of the present invention provide an isolated and purified FlaC protein of a Campylobacter strain substantially free from other proteins of the Campylobacter strain. The Campylobacter strain may be C. jejuni. 
The present invention further provides synthetic peptides corresponding to portions of the FlaC protein. Accordingly, in a further aspect of the invention, there is provided a synthetic peptide having no less than six amino acids and no more than 150 amino acids and containing an amino acid sequence corresponding to a portion only of a FlaC protein of a strain of Campylobacter or of a fragment or an analog of the FlaC protein.
In accordance with another aspect of the invention, an immunogenic composition is provided which comprises at least one active component selected from at least one nucleic acid molecule as provided herein, at least one recombinant protein as provided herein, at least one of the purified and isolated FlaC protein, as provided herein and at least one synthetic peptide as provided herein, and a pharmaceutically acceptable carrier therefor or vector therefor. The at least one active component produces an immune response when administered to a host.
The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to protect against diseases caused by bacterial pathogens that produce flagellin proteins. For such purpose, the compositions may be formulated as a microparticle, capsule, ISCOM or liposome preparation. Alternatively, the compositions may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. The immunogenic composition may comprise a plurality of active components to provide protection against disease caused by a plurality of species of flagellin protein producing bacteria.
The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant or at least one cytokine. Suitable adjuvants for use in the present invention include (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein. Advantageous combinations of adjuvants are described in copending U.S. patent applications Ser. No. 08/261,194 filed Jun. 16, 1994 and Ser. No. 08/483,856, filed Jun. 7, 1995, assigned to the assignee hereof and the disclosures of which are incorporated herein by reference thereto.
In accordance with another aspect of the invention, there is provided a method for inducing protection against infection or disease caused by Campylobacter or other bacteria that produce flagellin protein, comprising the step of administering to a susceptible host, which may be a primate, such as a human, an effective amount of the immunogenic composition as recited above.
In accordance with another aspect of the invention, an antiserum or antibody specific for the recombinant protein, the isolated and purified FlaC protein, synthetic peptide or the immunogenic composition, is provided.
In a further aspect, there is provided a live vector for delivery of FlaC protein to a host, comprising a vector containing the nucleic acid molecule as described above. The vector may be selected from Salmonella, BCG, adenovirus, poxvirus, vaccinia and poliovirus. The nucleic acid molecule may encode a fragment of the FlaC protein of a Campylobacter strain which is conserved among bacteria that produce the FlaC protein. Such vector may be included in an immunogenic composition provided herein.
The present invention further includes a method of determining the presence of nucleic acid encoding the FlaC protein of a strain of Campylobacter, in a sample, comprising the steps of: (a) contacting the sample with the nucleic acid molecule provided herein to produce duplexes comprising the nucleic acid molecule and any said nucleic acid molecule encoding the FlaC protein of Campylobacter present in the sample and specifically hybridizable therewith; and (b) determining production of the duplexes.
In an additional aspect, the present invention provides a method of determining the presence of a FlaC protein of a Campylobacter strain in a sample, comprising the steps of (a) immunizing a subject with the immunogenic composition provided herein to produce antibodies specific for the FlaC protein; (b) contacting the sample with the antibodies to produce complexes comprising FlaC protein of a Campylobacter strain present in the sample and the FlaC protein specific antibodies; and determining production of the complexes.
A further aspect of the present invention provides a diagnostic kit for determining the presence of nucleic acid encoding the FlaC protein of a strain of Campylobacter, in a sample, comprising (a) the nucleic acid molecule provided herein; (b) means for contacting the nucleic acid with the sample to produce duplexes comprising the nucleic acid molecule and any said nucleic acid present in the sample and hybridizable with the nucleic acid molecule; and (c) means for determining production of the duplexes.
In another aspect of the present invention, there is provided a diagnostic kit for detecting the presence of a FlaC protein of a Campylobacter strain in a sample, comprising (a) a FlaC protein specific antibody to the immunogenic composition provided herein; (b) means for contacting the antibody with the sample to produce a complex comprising said FlaC protein and the antibody; and (c) means for determining production of the complex.
The invention further includes the use of the nucleic acid molecules and proteins provided herein as medicines. The invention additionally includes the use of the nucleic acid molecules and proteins provided herein in the manufacture of medicaments for protection against infection by strains of Campylobacter.
The purified and isolated DNA molecules comprising at least a portion coding for a FlaC protein of a species of Campylobacter typified by the embodiments described herein are advantageous as:
nucleic acid probes for the specific identification of Campylobacter strains in vitro or in vivo.
the products encoded by the DNA molecules are useful as diagnostic reagents, antigens for the production of Campylobacter-specific antisera, for vaccination against the diseases caused by species of Campylobacter and (for example) detecting infection by Campylobacter.
peptides corresponding to portions of the FlaC protein as typified by the embodiments described herein are advantageous as diagnostic reagents, antigens for the production of Campylobacter-specific antisera, for vaccination against the diseases caused by species of Campylobacter and (for example) for detecting infection by Campylobacter.